Synthesis, Pairing, and Cellular Uptake Properties of C(6

May 2, 2012 - We wished to address this issue by preparing a prodrug of tc-DNA containing a metabolically labile group at C(6′) that promotes cellul...
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Synthesis, Pairing, and Cellular Uptake Properties of C(6′)Functionalized Tricyclo-DNA Jory Lietard and Christian J. Leumann* Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland S Supporting Information *

ABSTRACT: Tricyclo-DNA (tc-DNA) is a promising candidate for oligonucleotide-based therapeutic applications exhibiting increased affinity to RNA and increased resistance to nucleases. However, as many other oligonucleotide analogs, tc-DNA does not readily cross cell membranes. We wished to address this issue by preparing a prodrug of tc-DNA containing a metabolically labile group at C(6′) that promotes cellular uptake. Two monomeric nucleoside building blocks bearing an ester function at C(6′) (tcee-T and tchd-T) were synthesized starting from a known C(6′) functionalized bicyclic sugar unit to which the cyclopropane ring was introduced via carbene addition. NIS-mediated nucleosidation of the corresponding glycal with in situ persilylated thymine afforded the β-iodonucleoside exclusively that was dehalogenated via radical reduction. Diversity in the ester function was obtained by hydrolysis and reesterification. The two nucleosides were subsequently incorporated into DNA or tc-DNA by standard phosphoramidite chemistry. The reactivity of the ester function during oligonucleotide deprotection was explored and the corresponding C(6′) amide, carboxylic acid, or unchanged ester functions were obtained, depending on the deprotection conditions. Compared to unmodified DNA, these tc-DNA derivatives increased the stability of duplexes investigated with ΔTm/mod of +0.4 to +2.0 °C. The only destabilizing residue was tchd-T, most likely due to self-aggregation of the lipophilic side chains in the single stranded oligonucleotide. A decamer containing five tchd-T residues was readily taken up by HeLa and HEK 293T cells without the use of a transfection agent.

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INTRODUCTION Control of translation by oligonucleotides was first described more than three decades ago1 and has since evolved into powerful therapeutic strategies2,3 among which the antisense, the siRNA, and the anti micro-RNA approaches are most promising.4,5 However, the barriers to the development of nucleic acids as therapeutics are manifold and delivery into cells is often recognized as being the most challenging one.6 Other important parameters include resistance toward nuclease degradation and affinity for the target RNA sequence. Chemical modification has emerged as a powerful tool to tackle these issues.2 In particular, nucleoside analogues with restricted backbone flexibility have been shown to increase the affinity toward complementary RNA and improve the stability of the corresponding oligonucleotides.7 Such conformationally constrained nucleic acid structures include hexitol nucleic acids (HNA),8 locked nucleic acids (LNA),9,10 and bicyclo- and tricyclo-DNA (Figure 1).11−13 Tricyclo-DNA (tc-DNA) is an RNA structural mimic which binds to complementary RNA with high affinity and high selectivity.14 Furthermore, it is stable in serum, does not elicit RNaseH cleavage and has recently been evaluated as antisense and siRNA agent with promising results.15−17 However, like most natural or modified oligonucleotides, tc-DNA also suffers from poor cellular uptake and thus relies on the use of transfection agents to help crossing of the cellular membrane. © 2012 American Chemical Society

Figure 1. Chemical structures of selected derivatives of the bicycloand tricyclo-DNA family.

Transfection agents such as cationic lipids or polymers are perhaps the most known carriers for oligonucleotide delivery, Received: March 29, 2012 Published: May 2, 2012 4566

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Scheme 1. Synthesis of Tricyclo Compounds 3 and 4

Scheme 2. Synthesis of tcee-T Phosphoramidite 10

In this work, we set out to synthesize “pro tricyclooligonucleotides” bearing an ester function at position C(6′). This position was selected since it can easily be synthetically manipulated by virtue of the neighboring C(5′)-oxo function. Moreover, this position is expected to be sufficiently distant from the nucleobase to exclude interference with base pairing.

but their inherent toxicity has prevented their use in vivo.18 However, they represent only a fraction of a much broader arsenal of strategies for improving the cellular uptake of oligonucleotides. Such strategies include viral delivery methods,19 physical and mechanical techniques,20 and conjugation with gold nanoparticles21 or with arginine-rich or cellpenetrating peptides.22 Although they have been shown to improve the uptake of nucleic acids, limitations such as toxicity and immunogenicity have challenged their potential as universal delivery systems. Therefore, important research efforts were devoted to bypass the use of delivery agents. Cationic or zwitterionic oligonucleotides have been shown to promote cellular uptake in selected cases.23,24 In this context, we have recently reported on bicyclo-DNA carrying a lysine moiety able to cross the cell membrane without aid of transfection agents.25 Bioconjugation to cholesterol or long aliphatic chains has also proven successful in carrier-free approaches.26,27 Recently, LNA gapmers with phosphorothioates linkages have been evaluated in free cellular uptake through a process called gymnosis which requires higher oligonucleotide concentrations in the growth medium.28 Another promising concept consists in temporarily masking the negative charge of the phosphates and has been coined the “pro-oligonucleotide approach” in analogy to the prodrug approach.29,30 The masked, charge neutral oligonucleotide can more easily penetrate into the intracellular compartment where endogenous enzymes convert the pro-oligonucleotide into the biologically active nucleic acid. Variations of this theme include modification of the O(2′) position of ribonucleotides to attach biolabile groups in order to improve the cellular uptake of RNA.31,32

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RESULTS AND DISCUSSION

Synthesis of tcee-T and tchd-T Building Blocks 10 and 15. The preparation of tcee-T and tchd-T phosphoramidites 10 and 15 started from the already known ketone 1 (mixture of four diastereoisomers), which was previously used in the synthesis of C(6′)-alkylated bicyclo nucleosides.33 Abstraction of the α-hydrogen of the ketone with LiHMDS followed by subsequent trapping of the enolate with TBS-Cl yielded silylenol ethers 2 which were separated into the pure anomeric forms (Scheme 1). The introduction of the cyclopropane ring into 2 was first envisaged via the Simmons−Smith procedure (CH2I2, Ag/Zn couple) but resulted in very low yields and several undesired side products. When the Ag/Zn couple was exchanged for ZnEt2 (Furukawa’s protocol34), the reaction proceeded smoothly and in good yields. However, while cyclopropanation of 2β afforded tricyclo compound 3β as the only isomer, the same conditions applied to 2α led to a mixture of two diastereoisomers that could easily be separated. The major isomer 4α (62%) resulted from reaction at the concave (endo) face of the bicyclic structure, while the desired isomer 3α with exo configuration of the cyclopropane ring was only obtained in 30% yield. The stereochemical outcome of the reaction with 2β can be explained by the directing effect of the homoallylic hydroxy group and steric hindrance at the alternative endo face.35 In 2α, the methoxy substituent might 4567

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Scheme 3. Synthesis of tchd-T Phosphoramidite 15

Figure 2. X-ray structures of nucleoside: 8A (left), and 8B (right).

With nucleoside 8 in hand, access to the phosphoramidite 10 was straightforward. Tritylation of the 5′-OH group with DMTr-Cl afforded intermediate 9 which was subsequently phosphitylated under standard conditions to give phosphoramidite 10. To obtain the long-chain alkyl ester derivative tchd-T, ester 7 was first saponified to afford the 3′-desilylated carboxylic acid 11 in good yield, which was used in the next step without further purification (Scheme 3). Condensation of 11 with 1hexadecanol mediated by EDC·HCl and DMAP, followed by desilylation with HF·pyridine led to the free nucleoside 13 in 65% yield over two steps. Tritylation of O(5′) and phosphitylation of O(3′) to give 14 and 15, respectively, proceeded under similar conditions as for building block 10 but required somewhat longer reaction times. X-ray Structure of Nucleoside 8. To gain information on the conformational preferences of the new tricyclonucleoside derivatives, the solid-state structure of nucleoside 8 was solved by X-ray crystallography. The asymmetric unit contains two symmetry-unrelated molecules 8A and 8B (Figure 2). The main structural difference between 8A and 8B lies in the furanose substructure which belongs to the C(2′)-exo (2E) conformation in 8A and to the O(4′)-endo (0E) conformation in 8B, with calculated pseudorotation phase angles (P) of 342.8° (8A) and 85.2° (8B), respectively. Although the glycosidic torsion angles χ are slightly different (Table 1), both structures have the thymine moiety in the anti conformation. In the asymmetric unit, there exists a intricate pattern of intermolecular H-bonds as well as stacking contacts of the bases which probably accounts for the differences in χ

engage in hydrogen bonding to the tertiary hydroxy group preventing it from coordinating to the reactive metal species, thus giving rise to a mixture of isomers. Furthermore, the ester function at C(6′) might also play a directing role in the cyclopropanation reaction.36 The configuration at the cyclopropane ring was assigned by 1H NMR NOE, 1H−1H COSY, and 2D NOESY experiments (see the Supporting Information). For the nucleosidation step, we considered the Niodosuccinimide induced addition of persilylated thymine to the corresponding glycal 5 (Scheme 2). Indeed, this strategy has been successfully applied to unmodified tricyclo pyrimidine nucleosides, yielding only β-nucleosides.37 Therefore, the combined anomers of 3 were converted into glycal 5 by treatment with TMSOTf, affording only one product and thereby verifying the cyclopropyl ring configuration (Scheme 2). The crude, 3′-silylated glycal 5 was then subjected to a NISmediated nucleosidation with persilylated thymine which pleasingly yielded only the β-nucleoside 6. Without further purification, the C(2′)-iodonucleoside 6 was subjected to radical reduction with Bu3SnH and AIBN, providing nucleoside 7 in 67% yield over three steps. Removal of the O(5′)- and O(3′)-silyl protecting groups with HF·pyridine gave compound 8, the relative configuration at the anomeric center of which was assessed by 1H NMR difference NOE experiments. Irradiation of H(1′) led to strongly enhanced signals at the H(4′) (4%) and H(2′) (5.9%) (see the Supporting Information). These observations confirmed the β-configuration at C(1′). The structure of nucleoside 8 was also confirmed independently by X-ray crystallography. 4568

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required in order to achieve coupling efficiencies of 94% for tcee-T 10 and 97% for tchd-T 15, as judged from the trityl assay. A set of conditions for deprotection and detachment from solid support has been elaborated that either allows to conserve the ester functions in the oligonucleotides or to transform them into amide or carboxy functions (Scheme 4).33 Treatment with concd NH3 (55 °C, 16 h) converted all ethyl ester groups into the corresponding amide functions yielding oligonucleotides ON 1, 3, 5, 7−9 (Table 2). Treatment with potassium hydroxide led to hydrolysis of the ester function (→ ON 10), while deprotection with 25% benzylamine left the ester groups untouched (→ ON 11). For oligonucleotides containing tchd-T units we selected ethanolic ammonia (concd NH3/EtOH 1:3) as a deprotection mixture. While after 24 h at 40 °C no trace of aminolysis of the ester function was observed by ESI−-MS for singly modified sequences (ON 2 and 4), some aminolysis was observed with ON 6 containing two hexadecyl ester moieties. Deprotection in concd NH3/EtOH 1:3 at 40 °C for only 2 h was sufficient to produce the oligothymidylates containing five tcee-T or tchd-T units (ON 12 and 13). Indeed, no hydrolyzed ester in the crude material was detected by mass spectrometry. All oligonucleotides were purified by ion-exchange (IE) HPLC using standard methods. Purification of ON 13 bearing five C16 chains, however, was not as straightforward and required the use of 1 M NaClO4 as eluent and organic cosolvents. Under these conditions, ON 13 eluted, somewhat surprisingly, as a broad peak with multiple shoulders, most likely arising from oligonucleotide aggregates. Reversed-phase HPLC on C4 or C18 columns was impossible due to very strong retention of the oligonucleotide on the solid phase. After purification and subsequent desalting, the integrity of all oligonucleotides was routinely verified either by ESI− or

Table 1. Selected Torsion Angles and Furanose Puckers of Nucleoside 8 in Comparison to bc-, tc-, and Natural Deoxynucleosides nucleoside

furanose pucker

γ (deg)

δ (deg)

χ (deg)

8A 8B tc-dAa bc-Tb dNc

C(2′)-exo O(4′)-endo C(2′)-exo C(1′)-exo C(2′)-endo

149.9 158.3 152 149.3 57

100.8 99.4 107 133.5 122

−170 −125.9 −164 −107.6 −119

a

Conformation of a tricyclodeoxyadenosine unit in a DNA duplex (ref ). bData taken from ref 11. cAverage deoxynucleotide conformation in B-DNA (ref 39).

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and P values. In both molecules 8A and 8B the torsion angle γ falls into the antiperiplanar range, which is consistent with the values found for unmodified members of the bc-DNA and tcDNA families.11,38 In addition, the torsion angle δ is in a +ac orientation as observed in tc-DNA residues. Summarizing all the structural features, it appears that the addition of an alkyl group at position C(6′) does not significantly change the intrinsic conformational preferences of the tricyclic core unit. Oligonucleotide Synthesis and Deprotection. The previously prepared tcee-T and tchd-T phosphoramidites 10 and 15 were incorporated into a series of mixed-base decamer oligodeoxynucleotides as single or double substitutions by standard solid-phase synthesis on a 1.3 μmol scale (ON 1−11, Table 2). The modified units were introduced into either natural DNA (ON 1−6) or tc-DNA (ON 7−11). Additionally, decaoligothymidylates containing five alternating tcee-T, tchd-T, or tc-T substitutions and labeled with 6-carboxyfluorescein (FAM) at their 3′-end were prepared for cellular uptake experiments (ON 12−14). An increased concentration (0.15 M in CH3CN) and an extended coupling time of 12 min were

Table 2. Oligonucleotides Prepared with tcee-T and tchd-T Amidites 10 and 15a

a

Capital letters: natural DNA nucleotides. Capital italic letters: 6′-unfunctionalized tricyclodeoxynucleotides tc-A, tc-C, tc-G, tc-T (structure, see Figure 1, top right). Lowercase letter: 6′-functionalized tc-T units. bFAM = 6-carboxyfluorescein. 4569

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Scheme 4a

complement, ON 5 exhibits again stabilization of the hybrid duplex with Tm‘s comparable to those of single-modified oligonucleotides. This is also a feature found for unmodified tcunits in the same sequence context14 and confirms again that the grafting of a substituent at C(6′) on the tricyclo scaffold does not affect the pairing efficiency. On the other hand, upon replacement of an ethyl by a hexadecyl group, all duplexes were destabilized (Table 3). Moreover, this destabilization was particularly pronounced with DNA as a complement, where ΔTms of −9.0 and −8.0 °C were measured with ON 2 and 4, respectively. When paired with complementary RNA, oligonucleotides containing one tchd-T unit were less destabilizing (−5.2 and −4.7 °C). Such a loss in affinity was cumulative, as a double-modified oligonucleotide (ON 6) gave Tms of about half of that of the control (20.1 °C vs 44.9 °C; 23.1 °C vs 43.6 °C). Furthermore, this destabilization was associated with a lower hyperchromicity upon duplex melting, suggesting imperfect base-pairing (see the Supporting Information). UV−melting experiments were also performed in the series of decathymidylates alternately substituted with either tcee-T, tchd-T, or tc-T units and carrying a 3′-FAM label (Table 4). Table 4. Tm Data (°C) from UV−melting Curves (260 nm) of Modified Decathymidylates Sequences with Complementary DNA and RNAa

Oligonucleotide deprotection conditions: (a) concd NH3, 55 °C, 16 h; (b) 25% benzylamine in MeOH/H2O 1:2, 65 °C, 8 h; (c) 0.1 M KOH, 55 °C, overnight.

a

MALDI-TOF mass spectrometry (see the Supporting Information). Hybridization Properties of Substituted Oligodeoxynucleotides. The affinities of the modified oligodeoxynucleotides to their DNA and RNA complements was assessed by UV melting curves at 260 nm in standard saline buffer (10 mM Na2HPO4, 150 mM NaCl, pH 7.0) (Table 3). Introduction of a

ON ON ON ON ON ON

1 2 3 4 5 6

Tm (°C) vs DNA (ΔTm/mod)b 45.7 37.0 45.3 38.0 42.9 20.1

(+0.8) (−9.0) (+0.4) (−8.0) (−1.0) (−13.0)

Tm (°C) vs RNA (ΔTm/mod)c

ON 12 ON 13 ON 14

18.3 (−1.1) n.d. 19.2 (−1.0)

20.6 (−0.2) n.d. 19.9 (−0.3)

Conditions: 2 μM single strands in 150 mM NaCl and 10 mM NaH2PO4 at pH 7.0. bTm of the FAM-labeled unmodified duplex: 24.0 °C; cTm of the FAM-labeled unmodified duplex: 21.6 °C. n.d.: not detected.

Analysis of the Tm data revealed that discontinuous tc-T incorporations (ON 14) were destabilizing when paired to dA10 and rA10 as a complement (−1.0 and −0.3 °C/mod, respectively), a result that was observed similarly also for ON 12 containing five alternative tcee-T units. However, the presence of five tchd-T units (ON 13) made the determination of a Tm value impossible. Indeed, no sigmoidal curves and thus no duplex formation was observed upon melting of ON 13·with cDNA or RNA (see the Supporting Information). We hypothesize that this is the result of a competition between base pairing and hydrophobic interactions of the C16 side chains in ON 13, organizing it in structures such as micelles or vesicles where the oligonucleotide is less accessible for hybridization. Hybridization Properties of Substituted Tricyclooligonucleotides. The tc-T derivatives were also incorporated into tc-oligonucleotides to study the influence of a modified tc-unit on the pairing properties of tc-DNA (Table 5). UV−melting curve analysis showed that the strong stabilization brought about by the fully modified tc-DNA (+13.5 °C and +20.5 °C with cDNA and RNA, respectively) is conserved upon incorporation of the functionalized tc-units, with ΔTms ranging from −1.6 to +2.0 °C overall. This indicates that short alkyl chains at position C(6′) do not interfere with the pairing properties of tc-DNA, suggesting again a smooth accommodation of all substituents in the duplexes. The amide and ethyl

Tm (°C) vs RNA (ΔTm/mod)c 45.7 38.4 45.6 38.9 46.1 23.1

Tm (°C) vs DNA (ΔTm/mod)b

a

Table 3. Tm data (°C) from UV−melting Curves (260 nm) of Modified Decamer Sequences with Complementary DNA and RNAa entry

entry

(+2.1) (−5.2) (+2.0) (−4.7) (+1.3) (−10.2)

Conditions: 2 μM single strands in 150 mM NaCl and 10 mM NaH2PO4 at pH 7.0. bTm of the unmodified duplex: 44.9 °C; cTm of the unmodified duplex: 43.6 °C a

single tc-T unit bearing an amide group at position C(6′) (ON 1 and 3) led to an increase in Tm of +0.4 to +0.8 °C with a DNA complement and to +2.0 °C with an RNA complement. This clearly indicates that the amide substituent at C(6′) does not perturb duplex formation. Furthermore, the degree of stabilization is comparable to the single incorporation of an unmodified tc-T unit (−0.4 °C) in the same sequence.14 Two modifications separated by a natural dG unit (ON 5) slightly decreased the Tm of a DNA duplex when compared to the unmodified duplex (−1.0 °C/mod.). However, with RNA as a 4570

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unmodified dT10 with a fluorescein label as a control. The experiments were performed in pure water and in standard saline buffer (Figure 3). In water, low emission of fluorescence was obtained for all FAM-labeled oligonucleotides ON 12−14, suggesting a substantial degree of self-association under these conditions (Figure 3A). Upon addition of the detergent Triton x-100 above its critical micelle concentration, a large restoration of fluorescence emission was observed for ON 12 and 14 and dT10-FAM. However, the increase of fluorescence emission for ON 13 was limited (Figure 3B). In standard saline, the intensity of fluorescence emission for ON 12 and 14 and dT10FAM reached a maximum and did not vary upon addition of Triton x-100 (Figures 3C,D). On the other hand, under these conditions, a substantial quenching of fluorescence for ON 13 was still detected and was only partially restored after addition of Triton x-100. Taken together, these results indicate that ON 13 self-associates even in buffered saline. The limited restoration of fluorescence emission for ON 13 after addition of a detergent implies that self-quenching is not entirely eliminated. It is therefore reasonable that this amphiphilic oligonucleotide still exists in an aggregated state even in the presence of large quantities of a detergent. Cellular Uptake. The previously prepared fluoresceinlabeled oligonucleotides were then evaluated for their ability to cross the cellular membrane in the absence of a transfection agent. Two human cell lines, HeLa and HEK 293T, were transfected with ON 12, 13 and 14 at 10 μM concentration each, and the unmodified dT10-FAM was used as a control. After 2 days of incubation at 37 °C, the cells were fixed and analyzed by confocal fluorescence microscopy (Figure 4). No visible internalization of the control oligonucleotide dT10-FAM was detected in any cell line. Similarly, the decathymidylates modified with either tc-T or tcee-T (ON 12 and 14) were not

Table 5. Tm Data (°C) from UV−melting Curves (260 nm) of Modified Decamer Sequences in the Context of a tc-DNA Backbone with Complementary DNA and RNAa entry ON ON ON ON ON

7 8 9 10 11

Tm (°C) vs DNA (ΔTm/mod)b Tm (°C) vs RNA (ΔTm/mod)c 57.8 57.1 57.3 56.9 57.2

(−0.7) (−1.4) (−0.6) (−0.8) (−0.6)

65.0 64.4 63.9 66.3 63.9

(+0.7) (+0.1) (−0.2) (+1.0) (−0.2)

Conditions: 2 μM single strands in 150 mM NaCl and 10 mM NaH2PO4 at pH 7.0. bTm of fully modified tricyclo-DNA/DNA duplex: 58.5 °C; cTm of fully modified tricyclo-DNA/RNA duplex: 64.3 °C. a

ester groups were both found to have negligible effects on the thermal stability (ON 9 and 11). Interestingly, additional carboxylate groups (as in ON 10) led to an increase in Tm with RNA as a complement, implying that additional negative charges do not perturb the pairing affinity of tc-DNA. This is an important observation as according to the prodrug approach the carboxylic acid function is expected to be the functional group after enzymatic ester hydrolysis. It therefore appears that ester hydrolysis will be required to functionally activate the oligonucleotide. Fluorescence Emission Measurements. The analysis of the UV−melting curves of ON 13 led us to propose that selfassociation of the single strands via their hydrophobic hexadecyl ester functions might occur in water. Consequently, this should result in significant self-quenching of fluorescein fluorescence by proximity effects.40 We measured the emission of fluorescence of all FAM-labeled oligonucleotides after excitation at 480 nm. In addition, we used the corresponding

Figure 3. Fluorescence emission spectra of FAM-labeled oligonucleotides: (A) in H2O; (B) in H2O after addition of 0.36 mM Triton x-100; (C) in 150 mM NaCl, 10 mM NaH2PO4, pH 7.0; (D) in 150 mM NaCl, 10 mM NaH2PO4, pH 7.0 after addition of 0.36 mM Triton x-100. Conditions: 2 μM single strand, excitation at 480 nm, 20 °C. 4571

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Figure 4. Fluorescence microscopy analysis of HeLa (top row) and HEK 293T cells (bottom row) treated with: (A, E) dT10-FAM; (B, F) ON 12, (C, G) ON 14, (D, H) ON 13. Conditions: 10 μM ODN concentration, 2 days incubation at 37 °C, cells are then fixed with 3.7% paraformaldehyde and their nuclei stained with DAPI (blue channel).

releasing the corresponding carboxylic acid which was shown to increase DNA and RNA affinity. Furthermore, the cellular uptake of oligonucleotides containing either tc-T, tcee-T, or tchd-T units showed that a C16 side chain is capable of helping to cross the cellular membrane of two different cell lines. Fluorescence microscopy revealed a rather uniform cytosolic distribution of ON 13 together with surface-bound material. The exact mechanism by which ON 13 enters cells remains elusive at this point. Taken together, these results represent an important step toward the preparation of a tc-DNA prodrug. Further studies on the enzymatic processing of the hexadecyl ester and preliminary tests on the antisense activities of tcoligonucleotides containing tchd-T units are underway.

able to penetrate through the cellular membrane. On the other hand, ON 13 was readily taken up (Figure 4D,H) in both cell lines, thus highlighting the role of the side chains attached to the tricyclo skeleton. The distribution pattern was mainly cytoplasmic with a uniform rather than a granular localization. In HeLa cells, fluorescence was also visible on the contours of the cells, suggesting that some material remained associated with the cell membrane. In contrast, the contours of HEK cells were not fluorescent and ON 13 was more homogeneously distributed throughout the cytosol. These observations raise questions about the mechanism of internalization and the subsequent trafficking. Indeed, the uniform cytosolic distribution implies that if endocytosis was the pathway for uptake, then most of the material has leaked out of the endosomal compartment. Furthermore, the presence of membrane-bound oligonucleotide suggests that endocytosis may not be the only route of internalization. As an alternative pathway, the fusion of ON 13 with the cell surface can be assumed which has previously been shown to occur with cationic lipids as delivery agents.41 Clearly, additional experiments will be required in order to identify the mechanism of uptake for ON 13.

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EXPERIMENTAL SECTION

General Methods. All reactions were performed under Ar and in dried glassware. Anhydrous solvents for reactions were obtained by filtration through activated alumina or by storage over molecular sieves (4 Å). Column chromatography (CC) was performed on silica gel with an average particle size of 40 μm. All solvents for column chromatography were of technical grade and distilled prior to use. Thin-layer chromatography was performed on silica gel plates. Visualization was achieved either under UV light or by dipping in staining solution [CerIV-sulfate (10.5 g), phosphormolybdenic acid (21 g), conc. H2SO4 (60 mL), H2O (900 mL) or p-anisaldehyde (3.7 mL), concd H2SO4 (5 mL), glacial acetic acid (1.5 mL), ethanol (135 mL)] followed by heating with a heat gun. NMR spectra were recorded at 400 or 300 MHz field width (1H) in either CDCl3 or CD3OD. δ in ppm relative to residual undeuterated solvent [CHCl3: 7.26 ppm (1H) and 77.0 ppm (13C); CHD2OD: 3.35 ppm (1H) and 49.3 ppm (13C)], J in Hz. Signal assignments are based on DEPT and on 1H−1H and 1 H−13C correlation experiments (COSY/HSQC). High-resolution mass spectra were recorded on an ion-trap instrument in the ESI+ mode. (1S,5S)-8-[(tert-Butyldimethylsilyl)oxy]-7-(ethoxycarbonyl)methyl-5-hydroxy-3-methoxy-2-oxybicyclo[3.3.0]oct-7-ene (2). To a solution of 1 (1.0 g, 3.87 mmol)33 in dry THF (39 mL) was slowly added LiHMDS (842 mg, 5.03 mmol) 0.75 M THF at −78 °C. A solution of TBDMS-Cl (875 mg, 5.81 mmol) and Et3N (0.22 mL, 1.54 mmol) in dry THF (14 mL) was added dropwise after 20 min. The resulting clear yellow solution was allowed to warm to rt after 50 min and was stirred for an additional 2 h. The mixture was then diluted with EtOAc (150 mL) and washed with satd NaHCO3 (2 × 125 mL) and brine (1 × 125 mL). The aqueous phase was extracted with EtOAc (2 × 400 mL), and the combined organic phases were dried over MgSO4, filtered, and evaporated. The crude orange oil was purified by CC with EtOAc/hexane (1:5 → 1:1), affording 2α (1.03 g, 72%) and 2β (211 mg, 15%) as colorless oils. Data for 2α: Rf = 0.51 (EtOAc/hexane 2:1); 1H NMR (300 MHz, CDCl3) δ 5.00 (d, J = 4.1 Hz, 1H, H−C(3)), 4.65 (s, 1H, H−C(1)), 4.09 (2q, J = 7.8, 4.0 Hz, 2H, CH3CH2O), 3.35 (s, 3H, MeO), 3.10 (s, 1H, OH), 3.02 (s, 2H, H−C(6)), 2.47 (d, J = 0.8 Hz, 2H, H−C(9)),

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CONCLUSIONS In this work, we have presented the synthesis of tc-T derivatives with functionalities connected to C(6′) of the tricyclic sugar structure with the aim of improving the cellular uptake of tcDNA. Two phosphoramidites, tcee-T 10 and tchd-T 15, were prepared in 8 and 10 steps, respectively, from ketone 1. The key synthetic steps are the β-selective NIS-mediated nucleosidation and hydrolysis/re-esterification that allows for the preparation of a variety of tc-T esters without altering the major part of the synthetic scheme. Both phosphoramidites were successfully incorporated into oligonucleotides and the reactivity of the ester group was investigated under various deprotection conditions. Hydrolysis and aminolysis afforded the corresponding carboxylic acid and amide forms, whereas mild ethanolic ammonia treatment left the ethyl and hexadecyl esters untouched in most cases. Amide, acid or ethyl ester substituted oligonucleotides led to a significant stabilization when paired to cDNA, and this effect was even more expressed with RNA as a complement. With tchd-T however, all duplexes were destabilized and it appears that there exists a competition between base pairing and self-aggregation of the long aliphatic chains as evidenced by UV-melting experiments and fluorescence emission spectroscopy. Nevertheless, tchd-T maintains its potential as a nucleoside analogue for oligonucleotide-based therapies. Indeed, the hexadecyl ester is expected to be hydrolyzed after internalization into cells, thus 4572

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2.19 (d, J = 13.4 Hz, 1H, H−C(4)), 2.01 (dd, J = 13.4, 4.1 Hz, 1H, H− C(4)), 1.22 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.93 (s, 9H, (CH3)3C−Si), 0.18 (s, 3H, (CH3)2Si), 0.14 (s, 3H, (CH3)2Si); 13C NMR (75 MHz, CDCl3) δ 171.1 (CO2Et), 147.5 (C(8)), 110.73 (C(7)), 105.5 (C(3)), 92.0 (C(1)), 83.7 (C(5)), 60.8 (CH3CH2O), 54.7 (MeO), 47.2 (C(4)), 40.9 (C(6)), 32.4 (C(9)), 25.92 ((CH3)3C−Si), 18.5 ((CH3)3C-Si), 14.5 (CH3CH2O), −3.9, −4.2 ((CH3)2Si); ESI+HRMS m/z calcd for C18H32O6NaSi ([M + Na]+) 395.1860, found 395.1847. Data for 2β: Rf = 0.29 (EtOAc/hexane 2:1); 1H NMR (300 MHz, CDCl3) δ 5.15 (dd, J = 5.4, 2.7 Hz, 1H, H−C(3)), 4.53 (s, 1H, H− C(1)), 4.10 (q, J = 7.1 Hz, 2H, CH3CH2O), 3.30 (s, 3H, MeO), 3.16 (d, J = 15.8, 1H, H−C(6)), 2.89 (dd, J = 15.7, 0.8 Hz, 1H, H−C(6)), 2.74 (d, J = 15.6 Hz, 1H, H−C(9)), 2.48 (d, J = 15.6 Hz, 1H, H− C(9)), 2.32 (dd, J = 13.6, 5.4 Hz, 1H, H−C(4)), 2.16 (dd, J = 13.6, 2.7 Hz, 1H, H−C(4)), 1.99 (s, 1H, OH), 1.23 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.94 (s, 9H, (CH3)3C−Si), 0.19, 0.18 (2s, 6H, (CH3)2Si). 13 C NMR (75 MHz, CDCl3) δ 171.2 (CO2Et), 148.3 (C(8)), 110.4 (C(7)), 106.5 (C(3)), 92.2 (C(1)), 84.3 (C(5)), 60.8 (CH3CH2O), 55.3 (MeO), 48.8 (C(4)), 45.2 (C(9)), 32.6 (C(6)), 25.8 ((CH3)3C− Si), 18.39 ((CH3)3C-Si), 14.5 (CH3CH2O), −3.9, −4.1 ((CH3)2Si); ESI+-HRMS m/z calcd for C18H32O6NaSi ([M + Na]+) 395.1860, found 395.1858. (1S,2R,4R,6S,8R)-2-[(tert-Butyldimethylsilyl)oxy]-4(ethoxycarbonyl)methyl-6-hydroxy-8-methoxy-9-oxytricyclo[4.3.01,6.02,4]nonane (3β). To a solution of 2β (0.35 mg, 0.95 mmol) in dry CH2Cl2 (10 mL) was added diethylzinc (1 M in hexane, 5.7 mL, 5.7 mmol) dropwise at 0 °C, followed by diiodomethane (0.76 mL, 9.5 mmol) after 15 min. A white precipitate formed, and the resulting suspension was allowed to warm to rt. After 5 h, the mixture was diluted with EtOAc (100 mL) and washed with satd NH4Cl (1 × 50 mL) and 10% Na2S2O3 (1 × 50 mL). The aqueous phases were combined and extracted with EtOAc (2 × 100 mL). The organic phases were dried over MgSO4, filtered, and evaporated. The crude dark yellow solid was purified by CC with EtOAc/hexane (1:5 → 1:3) to give the title compound 3β (0.30 g, 81%) as a colorless oil. Data for 3β: Rf = 0.43 (hexane/EtOAc 2:1); 1H NMR (300 MHz, CDCl3) δ 5.03 (d, J = 4.8 Hz, 1H, H−C(8)), 4.18−4.09 (m, 3H, CH3CH2O, H−C(1)), 3.31 (s, 3H, MeO), 2.55 (m, 2H, H−C(10), H−C(5)), 2.23 (m, 2H, H−C(7), H−C(10)), 2.11 (dd, J = 12.4, 4.9 Hz, 1H, H−C(7)), 1.92 (d, J = 13.7 Hz, 1H, H−C(5)), 1.24 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.88 (s, 9H, (CH3)3C−Si), 0.85−0.80 (m, 2H, H−C(3)), 0.18 (s, 3H, (CH3)2Si), 0.09 (s, 3H, (CH3)2Si); 1H NMR difference NOE (400 MHz, CDCl3) δ 5.03 (H−C(8)) → 3.31 (5.5%, O-Me), 2.11 (3.7%, H−C(7)), 0.85 (H−C(3)) → 4.12 (2.2%, H− C(1)), 2.23 (1.9%, H−C(10)), 1.92 (1.7%, H−C(5)); 13C NMR (75 MHz, CDCl3) δ 173.3 (CO2Et), 105.6 (C(8)), 91.9 (C(1)), 86.8 (C(6)), 68.2 (C(2)), 60.5 (CH3CH2O), 54.5 (MeO), 49.4, 49.2 (C(7), C(5)), 37.3 (C(10)), 30.7 (C(4)), 25.9 ((CH3)3C−Si), 22.3 (C(3)), 18.1 ((CH3)3C-Si), 14.2 (CH3CH2O), −3.57, −3.62 ((CH3)2Si); ESI+-HRMS m/z calcd for C19H35O6Si ([M + H]+) 387.2197, found 387.2205. (1S,2R,4R,6S,8S)-2-[(tert-Butyldimethylsilyl)oxy]-4[(ethoxycarbonyl)methyl]-6-hydroxy-8-methoxy-9oxytricyclo[4.3.01,6.02,4]nonane (3α) and (1S,2S,4S,6S,8S)-2[(tert-butyldimethylsilyl)oxy]-4-[(ethoxycarbonyl)methyl]-6hydroxy-8-methoxy-9-oxytricyclo[4.3.01,6.02,4]nonane (4α). To a heavily stirred solution of 2α (1.01 g, 2.68 mmol) in dry CH2Cl2 (45 mL) was slowly added diethylzinc (1 M in hexane, 16.1 mL, 16.1 mmol) at 5−10 °C. After fuming had ceased, diiodomethane (2.15 mL, 2.68 mmol) was added dropwise and the mixture allowed to reach rt. After being stirred overnight, the reaction mixture was diluted with EtOAc (300 mL) and washed with satd NH4Cl (1 × 200 mL) and 10% Na2S2O3 (1 × 200 mL). The aqueous phases were extracted with EtOAc (2 × 400 mL) and the combined organic phases dried over MgSO4, filtered, and evaporated. Purification of the resulting yellow oil by CC (hexane/EtOAc 5:1 → 2:1) afforded 3α (0.31 g, 30%) and 4α (0.65 g, 62%) both as colorless oils. Data for 3α: Rf = 0.58 (hexane/EtOAc 3:2); 1H NMR (400 MHz, CDCl3) δ 5.07 (dd, J = 5.3, 1.6 Hz, 1H, H−C(8)), 4.15−4.09 (m, 3H,

CH3CH2O, H−C(1)), 3.36 (s, 3H, MeO), 2.52 (d, J = 15.8 Hz, 1H, H−C(10)), 2.43 (dd, J = 13.8, 5.3 Hz, 1H, H−C(7)), 2.32 (d, J = 15.8 Hz, 1H, H−C(10)), 2.14 (dd, J = 13.8, 1.2 Hz, 1H, H−C(5)), 2.00 (dd, J = 14.0, 1.6 Hz, 1H, H−C(7)), 1.96 (d, J = 13.9 Hz, 1H, H− C(5)), 1.88 (s, 1H, O-H), 1.24 (t, J = 7.1 Hz, 3H, CH3CH2O), 1.02 (dd, J = 6.1, 0.8 Hz, 1H, H−C(3)), 0.89 (s, 9H, (CH3)3C−Si), 0.79 (dd, J = 6.1, 1.6 Hz, 1H, H−C(3)), 0.19 (s, 3H, (CH3)3C−Si), 0.13 (s, 3H, (CH3)3C−Si); 1H NMR difference NOE (400 MHz, CDCl3) δ 5.07 (H−C(8)) → 3.36 (4.7%, O-Me), 2.43 (4.8%, H−C(7)); 0.79 (Hβ-C(3)) → 4.10 (3.9%, H−C(1)), 1.96 (1.7%, H−C(5)), 0.85 (25%, Hα-C(3)), 0.85 (Hα-C(3)) → 2.32 (2.9%, H−C(10)), 0.79 (20.3%, Hα-C(3)); 13C NMR (100 MHz, CDCl3) δ 173.0 (CO2Et), 105.7 (C(8)), 89.5 (C(1)), 86.1 (C(6)), 66.7 (C(2)), 60.5 (CH3CH2O), 55.0 (MeO), 50.0 (C(7)), 46.9 (C(5)), 37.1 (C(10)), 29.9 (C(4)), 26.0 ((CH3)3C−Si), 22.9 (C(3)), 18.2 ((CH3)3C-Si), 14.5 (CH3CH2O), −3.4, −3.8 ((CH3)2Si); ESI+-HRMS m/z calcd for C19H34O6SiNa ([M + Na]+) 409.2017, found 409.2008. Data for 4α: Rf = 0.73 (hexane/EtOAc 3:2); 1H NMR (400 MHz, CDCl3) δ 5.10 (d, J = 4.2 Hz, 1H, H−C(8)), 4.70 (d, J = 1.5 Hz, 1H, H−C(1)), 4.12 (qd, J = 7.1, 1.6 Hz, 2H, CH3CH2O), 3.34 (s, 3H, MeO), 3.21 (s, 1H, O-H), 2.50 (d, J = 16.3 Hz, 1H, H−C(10)), 2.34 (d, J = 16.3 Hz, 1H, H−C(10)), 2.17−2.07 (m, 2H, H−C(5)), 1.95 (d, J = 13.9 Hz, 1H, H−C(7)), 1.79 (dd, J = 13.9, 4.3 Hz, 1H, H−C(7)), 1.24 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.85 (s, 9H, (CH3)3C−Si), 0.85− 0.82 (m, 1H, H−C(3)), 0.62 (d, J = 6.5 Hz, 1H, H−C(3)), 0.17 (s, 3H, (CH3)3C−Si), 0.08 (s, 3H, (CH3)3C−Si); 1H NMR difference NOE (400 MHz, CDCl3) δ 5.10 (H−C(8)) → 3.34 (5.7%, O-Me), 1.79 (3.7%, H−C(7)), 4.70 (H−C(1)) → 3.21 (2.7%, O-H); 0.62 (Hβ-C(3)) → 2.32 (2.9%, H−C(5)), 0.83 (19.7%, Hα-C(3)); 13C NMR (75 MHz, CDCl3) δ 173.0 (CO2Et), 106.9 (C(8)), 98.2 (C(1)), 87.1 (C(6)), 68.3 (C(2)), 60.6 (CH3CH2O), 54.5 (MeO), 46.6 (C(7)), 44.0 (C(10)), 38.0 (C(5)), 32.0 (C(4)), 25.9 ((CH3)3C−Si), 22.3 (C(3)), 18.0 ((CH3)3C-Si), 14.5 (CH3CH2O), −4.0, −4.3 ((CH3)2Si); ESI+-HRMS m/z calcd for C19H35O6Si ([M + H]+) 387.2197, found 387.2198. (1S,2R,4R,6S)-2-[(tert-Butyldimethylsilyl)oxy]-4[ (e t h ox yc ar bo ny l )m e t h yl ]- 6 -h yd rox y - 9- ox y tr i c yc lo [4.3.01,6.02,4]non-7-ene (5). To a solution of 3 (323 mg, 0.84 mmol) in dry CH2Cl2 (6.1 mL) was added 2,6-lutidine (0.485 mL, 4.18 mmol) at 0 °C, followed by TMSOTf (0.43 mL, 2.51 mmol) after15 min. After an additional 30 min at 0 °C, the resulting yellow solution was warmed to rt and stirred for another 2 h. The reaction was then quenched by the dropwise addition of satd NaHCO3 (10 mL) and diluted with EtOAc (30 mL). The organic phase was washed with satd NaHCO3 (2 × 30 mL), and the aqueous phase was extracted with EtOAc (3 × 60 mL). The combined organic phases were then dried over MgSO4, filtered, and concentrated to yield crude 5 (385 mg) that was used without further purification in the next step. Data for 5: Rf = 0.88 (EtOAc/hexane 2:1); 1H NMR (300 MHz, CDCl3) δ 6.30 (d, J = 2.7 Hz, 1H, H−C(8)), 5.05 (d, J = 2.7 Hz, 1H, H−C(7)), 4.45 (s, 1H, H−C(1)), 4.11 (q, J = 7.1 Hz, 3H, CH3CH2O), 2.47 (d, J = 15.8 Hz, 1H, H−C(5)), 2.31 (d, J = 15.8 Hz, 1H, H−C(5)), 2.17 (dd, J = 13.4, 1.7 Hz, 1H, H−C(10)), 2.02 (d, J = 13.3 Hz, 1H, H−C(10)), 1.23 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.90 (dd, J = 5.9, 1.8 Hz, 1H, H−C(3)), 0.87 (s, 9H, (CH3)3C−Si), 0.81 (d, J = 5.9 Hz, 1H, H−C(3)), 0.17 (s, 3H, ((CH3)2Si), 0.13 (s, 3H, ((CH3)2Si), 0.07 (s, 9H, ((CH3)3Si); 13C NMR (75 MHz, CDCl3) δ 173.0 (CO2Et), 146.4 (C(8)), 108.6 (C(7)), 95.0 (C(1)), 91.6 (C(6)), 67.3 (C(2)), 60.5 (CH3CH2O), 50.8 (C(5)), 37.1 (C(10)), 30.7 (C(4)), 26.0 ((CH3)3C−Si), 23.9 (C(3)), 18.2 ((CH3)3C-Si), 14.5 (CH3CH2O), 2.1 ((CH3)3Si), −3.5, −3.6 ((CH3)2Si); ESI+HRMS m/z calcd for C21H39O5Si2 ([M + H]+) 427.2331, found 427.2331. 1-[(3′S,5′R,6′R)-3′-O-Trimethylsilyl-5′-O-(tert-butyldimethylsilyl)-6′-[(ethoxycarbonyl)methyl]-2′-deoxy-2′-iodo-3′,5′-ethano-5′,6′-methano-β-D-ribofuranosyl]thymine (6). To a mixture of thymine (530 mg, 4.2 mmol) in dry CH2Cl2 (6 mL) was added BSA (1.53 mL, 6.3 mmol) at rt. It was left for 1 h until a fine suspension had formed. A solution of 5 (668 mg, 1.4 mmol) in dry CH2Cl2 (6 mL) was then slowly added. After being cooled to 0 °C, N4573

dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

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CD3OD) δ 174.4 (CO2Et), 166.7 (C(4)), 152.3 (C(2)), 138.4 (C(6)), 111.2 (C(5)), 92.8 (C(4′)), 88.0 (C(1′)), 85.2 (C(3′)), 66.6 (C(5′)), 61.7 (CH3CH2O), 49.2 (C(2′)), 47.8 (C(7′)), 37.2 (C(9′)), 31.2 (C(6′)), 22.4 (C(8′)), 14.7 (CH3CH2O), 12.6 (Me-C(5)); ESI+HRMS m/z calcd for C17H23O7N2 ([M + H]+) 367.1500, found 367.1507. 1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′[(ethoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′-methano-β-D-ribofuranosyl]thymine (9). To a colorless solution of 8 (257 mg, 0.7 mmol) in dry pyridine (3.4 mL) was added DMTr-Cl (548 mg, 1.6 mmol) in four equal portions over a time range of 2.5 h at rt. The resulting orange solution was stirred for 8.5 h before addition of another 0.5 eq. of DMTr-Cl. After reaction overnight the mixture was diluted with EtOAc (30 mL) and subsequently washed with satd NaHCO3 (2 × 25 mL). The aqueous phase was extracted with EtOAc (3 × 50 mL). The combined organic phases were dried over MgSO4, filtered and evaporated. The resulting orange foam was purified by CC with EtOAc/hexane (1:1 → 4:1) containing 0.1% Et3N to give DMTr-protected nucleoside 9 (366 mg, 78%) as a white foam. Data for 9: Rf = 0.58 (EtOAc/EtOH 95:5); 1H NMR (300 MHz, CDCl3) δ 8.42 (br, 1H, H−N(3)), 8.21 (d, J = 1.0 Hz, 1H, H−C(6)), 7.44−7.41 (m, 2H, H-arom), 7.33 (dd, J = 8.9, 6.8 Hz, 1H, H-arom), 7.22−7.19 (m, 3H, H-arom), 6.77 (t, J = 8.9 Hz, 1H, H-arom), 5.83 (dd, 1H, H−C(1′)), 4.16 (m, 2H, CH3CH2O), 3.76, 3.75 (2s, 6H, MeO), 2.86 (d, J = 16.6 Hz, 1H, H−C(9′)), 2.51 (d, J = 16.6 Hz, 1H, H−C(9′)), 2.30 (dd, J = 11.4, 5.9 Hz, 2H, H−C(2′)), 2.19 (d, J = 14.2 Hz, 1H, H−C(7′)), 2.11 (s, 3H, Me-C(5)), 1.90−1.87 (m, 2H, H− C(7′), O-H), 1.21 (t, J = 7.1 Hz, 1H, CH3CH2O), 0.98 (d, J = 5.8 Hz, 1H, H−C(8′)), 0.73 (d, J = 6.4 Hz, 1H, H−C(8′)); 13C NMR (75 MHz, CDCl3) δ 172.8 (CO2Et), 164.0 (C(4)), 159.1 (MeO-C-arom), 150.3 (C(2)), 146.3, 136.9, 136.8 (C-arom), 136.7 (C(6)), 131.7, 131.6, 129.3, 127.7, 127.3, 113.0, 112.9 (CH-arom), 110.5 (C(5)), 91.3 (C(4′)), 86.4 (C(1′)), 88.1, 85.0 (C(3′)), C(Ph)3), 68.7 (C(5′)), 60.7 (CH3CH2O), 55.5 (MeO−DMTr), 49.4 (C(2′)), 47.5 (C(7′)), 36.7 (C(9′)), 31.4 (C(6′)), 22.7 (C(8′)), 14.5 (CH3CH2O), 12.6 (MeC(5)); ESI+-HRMS m/z calcd for C38H40O9N2Na ([M + Na]+) 691.2626, found 691.2640. 1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′[(ethoxycarbonyl)methyl]-3′-O-(2-cyanoethoxy)diisopropylaminophosphanyl-2′-deoxy-3′,5′-ethano-5′,6′methano-β-d-ribofuranosyl]thymine (10). To a solution of 9 (40 mg, 0.06 mmol) and N-ethyldiisopropylamine (0.04 mL, 0.24 mmol) in dry THF (0.4 mL) was carefully added 2-cyanoethyl N,Ndiisopropylchlorophosphoramidite (0.03 mL, 0.12 mmol) at rt. After the slightly yellow solution was stirred for 1 h, the mixture was diluted with EtOAc (20 mL) and washed with satd NaHCO3 (2 × 10 mL). The aqueous phase was extracted with EtOAc (3 × 20 mL), and the combined organic phases were dried over MgSO4. After filtration and evaporation, the residual oil was purified by CC (EtOAc/Hexane 1:1 → 4:1 + 0.1% Et3N) to give the title compound 10 (45 mg, 87%) as a white foam. Data for 10: Rf = 0.57, 0.63 (EtOAc/hexane 4:1); 1H NMR (300 MHz, CDCl3) δ 8.24 (br, 1H, H−N(3)), 8.17 (d, J = 1.2 Hz, 1H), 7.45−7.42 (m, 2H, H-arom), 7.33 (dd, J = 8.7, 5.8 Hz, 4H, H-arom), 7.23−7.21 (m, 3H, H-arom), 6.77 (dd, J = 8.9, 7.7 Hz, 4H, H-arom), 5.83 (dd, J = 6.8, 4.4 Hz, 1H, H−C(1′)), 4.18−4.09 (m, 2H, CH3CH2O), 3.78, 3.77, 3.76 (3s, 6H, MeO), 3.71−3.38 (m, 4H, OCH2CH2CN, (Me2CH)2N), 2.89−2.82 (2d, 1H, H−C(9′)), 2.72 (dd, J = 14.3, 6.9 Hz, 1H, H−C(2′)), 2.58−2.51 (m, 3H, OCH2CH2CN, H−C(9′)), 2.48−2.44 (m, 2H, H−C(7′), H−C(4′)), 2.32 (dd, J = 13.6, 4.9 Hz, 0.5H, H−C(2′)), 2.25 (dd, J = 14.1, 4.2 Hz, 0.5H, H−C(2′)), 2.10 (br, 3H, Me-C(5)), 2.05−1.99 (m, 1H,), 1.21, 1.20 (2t, J = 7.0 Hz, 3H, CH3CH2O), 1.10−1.05 (m, 12H, (Me2CH)2N), 0.95 (m, 1H, H−C(8′)), 0.85 (d, J = 6.5 Hz, 0.5H, H−C(8′)), 0.77 (d, J = 6.3 Hz, 0.5H, H−C(8′)); 13C NMR (75 MHz, CDCl3) δ 172.7, 172.5 (CO2Et), 164.1 (C(4)), 159.0 (MeO-C-arom), 150.2 (C(2)), 146.3, 146.2, 136.9, 136.8 (C-arom), 136.74, 136.66 (C(6)), 131.7, 131.6, 129.3, 127.7, 127.3, 127.2 (CH-arom), 117.77, 117.7 (CN), 113.0, 112.9 (CH-arom), 110.32, 110.26 (C(5)), 91.8, 91.2 (C(4′)), 88.3, 88.2, 88.1 (C(3′)), C(Ph)3), 87.2, 86.9 (C(1′)),

iodosuccinimide (504 mg, 2.24 mmol) was added portionwise over 1.5 h. The mixture was stirred for an additional 30 min at 0 °C and for 1.5 h at rt. The clear, brown solution was diluted with EtOAc (50 mL) and washed with satd NaHCO3 (2 × 50 mL) and satd Na2CO3 (1 × 50 mL). The aqueous phase was extracted with EtOAc (3 × 200 mL), and the combined organic phases were dried over MgSO4, filtered, and evaporated to give 1.2 g (>100%) of a viscous orange oil that was subjected to the next reaction without further purification. Data for 6: Rf = 0.4 (EtOAc/hexane 2:1); 1H NMR (300 MHz, CDCl3) δ 8.48 (br, 1H, H−N(3)), 7.69 (d, J = 1.2 Hz, 1H, H−C(6)), 6.35 (d, J = 5.9 Hz, 1H, H−C(1′)), 4.95 (d, J = 5.9 Hz, 1H, H−C(2′)), 4.30 (s, 1H, H−C(4′)), 4.11 (q, 2H, CH3CH2O), 2.56 (d, J = 16.9 Hz, 1H, H−C(7′)), 2.22 (d, J = 16.9 Hz, 1H, H−C(7′)), 1.96 (m, 2H, H− C(9′)), 1.90 (d, J = 1.1 Hz, 3H, Me-C(5)), 1.24 (t, J = 7.1, 0.8 Hz, 3H, CH3CH2O), 0.94 (d, J = 6.3 Hz, 1H, H−C(8′)), 0.85 (s, 10H, (CH3)3C−Si, H−C(8′)), 0.19 (s, 9H, (CH3)3Si), 0.12 (d, J = 0.8 Hz, 6H, (CH3)2Si); 13C NMR (75 MHz, CDCl3) δ 172.4 (CO2Et), 164.0 (C(4)), 150.6 (C(2)), 136.3 (C(6)), 110.7 (C(5)), 93.9 (C(1′)), 91.5 (C(4′)), 88.8 (C(3′)), 66.3 (C(5′)), 60.9 (CH3CH2O), 42.8 (C(9′)), 40.4 (C(2′)), 36.4 (C(7′)), 29.9 (C(6′)), 25.9 ((CH3)3C−Si), 25.7 (C(8′)), 18.1 ((CH3)3C-Si), 14.5 (CH3CH2O), 12.8 (Me-C(5)), 2.3 ((CH3)3Si), −3.5, −3.9 ((CH3)2Si); ESI+-HRMS m/z calcd for C26H43O7N2INaSi2 ([M + Na]+) 701.1546, found 701.1557. 1-[(3′S,5′R,6′R)-3′-O-Trimethylsilyl-5′-O-(tert-butyldimethylsilyl)-6′-[(ethoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′methano-β-D-ribofuranosyl]thymine (7). To an orange suspension of 6 (1.17 g, 1.4 mmol) and Bu3SnH (0.56 mL, 2.1 mmol) in dry toluene (12 mL) was added AIBN (115 mg, 0.7 mmol) at rt. The mixture was heated to 95 °C for 1 h. The yellow solution was then evaporated and the residue purified by CC with hexane/EtOAc (6:1 → 1:1) + 1% Et3N. Nucleoside 7 (515 mg, 67% from 3) was isolated as a white foam. Data for 7: Rf = 0.65 (EtOAc/hexane 1:1); 1H NMR (300 MHz, CDCl3) δ 8.58 (br, 1H, H−N(3)), 7.79 (d, J = 1.1 Hz, 1H, H−C(6)), 5.97 (dd, J = 5.3, 3.4 Hz, 1H, H−C(1′)), 4.26 (s, 1H, H−C(4′)), 4.11 (q, J = 7.1 Hz, 2H, CH3CH2O), 2.56 (m, 2H, H−C(2′)), 2.23−2.20 (m, 2H, H−C(9′)), 2.15 (d, J = 14.2 Hz, 1H, H−C(7′)), 1.91 (d, J = 0.9 Hz, 3H, Me-C(5)), 1.75 (d, J = 14.1 Hz, 1H, H−C(7′)), 1.23 (t, J = 7.1 Hz, 3H, CH3CH2O), 1.01 (d, J = 6.2 Hz, 1H, H−C(8′)), 0.90 (s, 9H, (CH3)3C−Si), 0.88−0.81 (m, 2H, H−C(8′)), 0.19 (s, 3H, (CH3)2Si), 0.14 (s, 3H, (CH3)2Si), 0.10 (s, 9H, (CH3)3Si); 13C NMR (100 MHz, CDCl3) δ 172.4 (CO2Et), 164.2 (C(4)), 150.2 (C(2)), 136.4 (C(6)), 109.6 (C(5)), 94.2 (C(4′)), 89.2 (C(1′)), 86.7 (C(3′)), 66.9 (C(5′)), 60.7 (CH3CH2O), 47.9 (C(2′)), 47.2 (C(7′)), 36.6 (C(9′)), 29.6 (C(6′)), 25.9 ((CH3)3C−Si), 21.7 (C(8′)), 18.1 ((CH3)3C-Si), 14.5 (CH3CH2O), 12.8 (Me-C(5)), 2.1 ((CH3)3Si), −3.6, −3.7 ((CH3)2Si); ESI+-HRMS m/z calcd for C26H45O7N2Si2 ([M + H]+) 553.2760, found 553.2769. 1-[(3′S,5′R,6′R)-6′-[(Ethoxycarbonyl)methyl]-2′-deoxy-3′,5′ethano-5′,6′-methano-β-D-ribofuranosyl]thymine (8). To a solution of 7 (510 mg, 0.92 mmol) in dry pyridine (6 mL) was carefully added HF (70% in pyridine, 0.34 mL, 18.4 mmol) at 0 °C. After 20 min of stirring, the reaction mixture was allowed to warm to rt. After a total of 32 h, the yellow reaction mixture was diluted with EtOAc (40 mL) and silica gel (4 g) was added. The mixture was concentrated, coevaporated with toluene (2 × 10 mL) and purified by CC (CH2Cl2/EtOH 95:5 to 9:1) to give 8 (290 mg, 86%) as white crystals. Data for 8: Rf = 0.31 (CH2Cl2/EtOH 9:1); 1H NMR (400 MHz, CD3OD) δ 7.94 (d, J = 1.2 Hz, 1H, H−C(6)), 6.16 (dd, J = 6.7, 5.7 Hz, 1H, H−C(1′)), 4.19−4.12 (m, 3H, CH3CH2O, H−C(4′)), 2.59 (d, J = 16.4 Hz, 1H, H−C(9′)), 2.49 (dd, J = 13.9, 6.8 Hz, 1H, H−C(2′)), 2.43−2.37 (m, 2H, H−C(2′), H−C(9′)), 2.15 (dd, J = 13.9, 1.4 Hz, 1H, H−C(7′)), 1.94 (d, J = 1.1 Hz, 3H, Me-C(5)), 1.92 (d, 1H, H− C(7′)), 1.26 (t, J = 7.1 Hz, 3H, CH3CH2O), 1.01 (d, J = 5.9 Hz, 1H, H−C(8′)), 0.82 (dd, J = 5.9, 1.6 Hz, 1H, H−C(8′)). 1H NMR difference NOE (400 MHz, CD3OD) δ 7.95 (H−C(6)) →6.17 (2.5%, H−C(1′)), 2.41 (2.9%, H−C(2′), H−C(9′)), 2.16 (1.2%, H−C(7′)), 1.95 (5%, Me-C(5)); 6.17 (H−C(1′)) →7.94 (2.1%, H−C(6)), 4.14 (4%, H−C(4′)), 2.49 (5.9%, H−C(2′)); 13C NMR (100 MHz, 4574

dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

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being stirred overnight, the mixture was heated to 45 °C for 4 h to complete conversion. The resulting turbid solution was diluted with EtOAc (20 mL) and the product adsorbed on silica gel (3 g). Evaporation followed by purification of the adsorbed material by CC (CH2Cl2/EtOH 9:1) afforded 13 (192 mg, 65% over two steps) as a white foam. Data for 13: Rf = 0.60 (CH2Cl2/EtOH 9:1); 1H NMR (300 MHz, MeOD) δ 7.93 (d, J = 1.1 Hz, 1H, H−C(6)), 6.15 (dd, J = 6.6, 5.7 Hz, 1H, H−C(1′)), 4.14 (s, 1H, H−C(4′)), 4.11 (td, J = 6.6, 1.6 Hz, 2H, CH2O), 2.59 (d, J = 16.3 Hz, 1H, H−C(9′)), 2.49 (dd, J = 13.9, 6.8 Hz, 1H, H−C(2′)), 2.44−2.35 (m, 2H, H−C(2′), H−C(9′)), 2.13 (dd, J = 13.9, 1.1 Hz, 1H, H−C(7′)), 1.94 (d, J = 0.9 Hz, 3H, Me-C(5)), 1.94−1.89 (m, 1H, H−C(7′)), 1.70−1.59 (m, 2H, CH2CH2O), 1.33− 1.29 (m, 26H, CH2-alk), 1.01 (d, J = 5.9 Hz, 1H, H−C(8′)), 0.90 (t, J = 6.7 Hz, 3H, CH3-alk), 0.82 (dd, J = 5.9, 1.4 Hz, 1H, H−C(8′)); 13C NMR (101 MHz, MeOD) δ 174.5 (CO2R), 166.8 (C(4)), 152.4 (C(2)), 138.4 (C(6)), 111.2 (C(5)), 92.8 (C(4′)), 88.0 (C(1′)), 85.1 (C(3′)), 66.7 (C(5′)), 65.9 (CH2O), 49.8, 49.6, 49.4, 49.3, 49.2, 49.0, 48.7, 48.5 (CHD2OD, C(2′)), 47.8 (C(7′)), 37.2 (C(9′)), 33.2 (C(6′)), 31.2, 31.0, 30.91, 30.89, 30.87, 30.81, 30.78, 30.6, 30.5 (CH2alk), 29.9 (CH2CH2O), 27.2, 23.9 (CH2-alk), 22.4 (C(8′)), 14.6 (CH3alk), 12.7 (Me-C(5)); ESI+-HRMS m/z calcd for C31H51O7N2 ([M + H]+) 563.3691, found 563.3678. 1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′[(hexadecoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′methano-β-D-ribofuranosyl]thymine (14). To a colorless solution of 13 (190 mg, 0.34 mmol) in dry pyridine (2.5 mL) was added DMTr-Cl (343 mg, 1.01 mmol) in four equal portions over 1.5 h. The resulting orange solution was stirred for 22 h before being diluted with EtOAc (30 mL). The organic phase was washed with satd NaHCO3 (2 × 20 mL) and the aqueous phase extracted with EtOAc (3 × 50 mL). The combined organic phases were dried over MgSO4, filtered, and concentrated. The crude orange foam was purified by CC (hexane/ EtOAc 1:1 + 0.1% Et3N, then hexane/EtOAc 1:4) to yield 14 (200 mg, 69%) as a slightly yellow foam. Data for 14: Rf = 0.48 (EtOAc/hexane 4:1); 1H NMR (300 MHz, CDCl3) δ 8.89 (s, 1H, H−N(3)), 8.24 (d, J = 0.8 Hz, 1H, H−C(6)), 7.42 (d, J = 9.1 Hz, 2H, H-arom), 7.32 (t, J = 8.3 Hz, 4H, H-arom), 7.23−7.12 (m, 3H, H-arom), 6.79−6.70 (m, 4H, H-arom), 5.84 (t, J = 6.1 Hz, 1H, H−C(1′)), 4.13−4.03 (m, 2H, CH2O), 3.73 (s, 3H, MeO), 3.71 (s, 3H, MeO), 2.88 (d, J = 16.7 Hz, 1H, H−C(9′)), 2.52 (d, J = 16.7 Hz, 1H, H−C(9′)), 2.46 (s, 1H, OH), 2.33 (dd, J = 14.0, 6.7 Hz, 1H, H−C(2′)), 2.29−2.18 (m, 3H, H−C(7′), H−C(4′)), 2.12 (s, 3H, Me-C(5)), 1.90 (d, J = 14.0 Hz, 1H, H−C(7′)), 1.61−1.50 (m, 2H, CH2CH2O), 1.29−1.15 (m, 26H, CH2-alk), 0.95 (d, J = 6.1 Hz, 1H, H−C(8′)), 0.86 (t, J = 6.7 Hz, 3H, CH3-alk), 0.73 (d, J = 6.3 Hz, 1H, H−C(8′)); 13C NMR (101 MHz, CDCl3) δ 173.0 (CO2R), 164.2 (C(4)), 159.1 (MeO-C-arom), 150.5 (C(2)), 146.4, 136.9 (C-arom), 136.7 (C(6)), 131.7, 131.6, 129.2, 127.6, 127.1, 112.9, 112.8 (CHarom), 110.8 (C(5)), 91.0 (C(4′)), 87.9 (C(3′)), 86.0 (C(1′)), 85.2 (C(Ph)3), 68.5 (C(5′)), 64.9 (CH2O), 55.4 (MeO), 49.5 (C(2′)), 47.4 (C(7′)), 36.6 (C(9′)), 32.1 (C(6′)), 31.3, 29.91, 29.87, 29.77, 29.75, 29.6, 29.4 (CH2-alk), 28.8 (CH2CH2O), 26.2, 22.9 (CH2-alk), 22.8 (C(8′)), 14.3 (CH3-alk), 12.6 (Me-C(5)); ESI+-HRMS m/z calcd for C52H68O9N2Na ([M + Na]+) 887.4817, found 887.4814. 1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′[(hexadecoxycarbonyl)methyl]-3′-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2′-deoxy-3′,5′-ethano-5′,6′methano-β-D-ribofuranosyl]thymine (15). To a colorless solution of N-ethyldiisopropylamine (0.16 mL, 0.93 mmol, 4 equiv) and nucleoside 15 (200 mg, 0.23 mmol) in 2.5 mL of dry THF (2.5 mL) was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.1 mL, 0.45 mmol) at rt. The resulting yellow solution was stirred for 1 h and gradually turned into a suspension. The reaction mixture was then diluted with EtOAc (40 mL) and washed with satd NaHCO3 (2 × 25 mL). The aqueous phase was extracted with EtOAc (3 × 50 mL), and the combined organic phases were dried over MgSO4, filtered, and evaporated. The crude yellow oil was purified by CC (hexane/EtOAc 1:1 +0.1% Et3N) to give 15 (220 mg, 89%, mixture of isomers) as a white foam.

68.5 (C(5′)), 60.7 (CH3CH2O), 58.1, 57.9 (JC,P = 15.5 Hz, OCH2CH2CN), 55.43, 55.40 (MeO−DMTr), 47.1, 47.0 (C(2′)), 45.2, 45.1 (C(7′)), 43.6, 43.5, 43.3 (JC,P = 12.7 Hz, (Me2CH)2N), 36.8, 36.7 (C(9′)), 31.33, 31.26, 24.7, 24.6, 24.5, 24.4 ((Me2CH)2N), 22.3 (C(8′)), 20.5, 20.4 (JC,P = 8.1 Hz, OCH2CH2CN), 14.5 (CH3CH2O), 12.7 (Me-C(5)). 31P NMR (122 MHz, CDCl3) δ 144.0, 142.3; ESI+HRMS m/z calcd for C47H58O10N4P ([M + H]+) 869.3885, found 869.3896. 1-[(3′S,5′R,6′R)-5′-O-(tert-Butyldimethylsilyl)-6′-(carboxymethyl)-2′-deoxy-3′,5′-ethano-5′,6′-methano-β-Dribofuranosyl]thymine (11). To a solution of 7 (520 mg, 0.94 mmol) in EtOH (2 mL) was added 4 N KOH (1.06 mL, 4.23 mmol) at rt. The resulting yellow solution was stirred for 2.5 h and was then diluted with H2O (20 mL). Neutralization was performed by 1 M KHSO4 (until pH 3 was reached, 12 mL). Extraction of the aqueous phase with CH2Cl2 (4 × 40 mL), followed by drying of the organic phase over MgSO4, filtration, and evaporation yielded 11 (391 mg, 92%) as a yellow foam that was used without further purification in the next step. Data for 11: Rf = 0.18 (CH2Cl2/EtOH 9:1); 1H NMR (300 MHz, MeOD) δ 7.91 (d, J = 1.0 Hz, 1H, H−C(6)), 6.06 (dd, J = 6.4, 3.4 Hz, 1H, H−C(1′)), 4.25 (s, 1H, H−C(4′)), 2.60 (dd, J = 13.9, 3.4 Hz, 1H, H−C(2′)), 2.49 (dd, J = 13.9, 6.5 Hz, 1H, H−C(2′)), 2.35 (q, J = 15.9 Hz, 2H, H−C(9′)), 1.96 (s, 2H, H−C(7′)), 1.90 (d, J = 0.9 Hz, 3H, Me-C(5)), 1.02 (d, J = 6.2 Hz, 1H, H−C(8′)), 0.94 (s, 9H, (CH3)3C− Si), 0.91 (d, J = 4.4 Hz, 1H, H−C(8′)), 0.23 (s, 3H, (CH3)2Si), 0.19 (s, 3H, (CH3)2Si); 13C NMR (75 MHz, MeOD) δ 176.0 (CO2H), 166.8 (C(4)), 152.3 (C(2)), 138.2 (C(6)), 110.5 (C(5)), 94.1 (C(4′)), 89.8 (C(1′)), 86.3 (C(3′)), 68.6 (C(5′)), 48.8 (C(2′)), 47.7 (C(7′)), 37.5 (C(9′)), 31.1 (C(6′)), 26.4 ((CH3)3C−Si), 23.4 (C(8′)), 19.0 ((CH3)3C-Si), 13.0 (Me-C(5)), −3.4, −3.6 ((CH3)2Si); ESI+-HRMS m/z calcd for C21H33O7N2Si ([M + H]+) 453.2052, found 453.2059. 1-[(3′S,5′R,6′R)-5′-O-(tert-Butyldimethylsilyl)-6′[(hexadecoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′methano-β-D-ribofuranosyl]thymine (12). To a slightly turbid solution of 11 (50 mg, 0.11 mmol) in dry CH2Cl2 (1.5 mL) was added 1-hexadecanol (32 mg, 0.13 mmol) followed by DMAP (7 mg, 0.056 mmol) at rt. The resulting mixture was cooled in an ice bath, and a solution of EDC·HCl (32 mg, 0.17 mmol) in dry CH2Cl2 (1.5 mL) was added dropwise. The mixture was allowed to warm to rt and was stirred for another 1 h. The clear solution was then diluted with EtOAc (25 mL) and washed with satd NaHCO3 (2 × 15 mL) and H2O (1 × 15 mL). The aqueous phase was extracted with EtOAc (3 × 45 mL), and the combined organic phases were dried over MgSO4, filtered, and concentrated. The crude material was used without further purification in the next step. For characterization, a sample of the crude material was purified by column chromatography (5% EtOH in CH2Cl2). Data for 12: Rf = 0.51 (CH2Cl2/EtOH 9:1); 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H, H−N(3)), 7.82 (d, J = 1.1 Hz, 1H, H−C(6′)), 6.03 (dd, J = 6.1, 3.1 Hz, 1H, H−C(1′)), 4.27 (s, 1H, H−C(4′)), 4.03 (t, J = 6.8 Hz, 2H, CH2O), 2.63 (dd, J = 14.0, 3.2 Hz, 1H, H−C(2′)), 2.56 (dd, J = 14.1, 6.2 Hz, 1H, H−C(2′)), 2.30 (q, J = 16.1 Hz, 2H, H−C(9′)), 2.13 (s, 1H, OH), 2.04−1.90 (m, 2H, H−C(7′)), 1.91 (d, J = 0.8 Hz, 3H, Me-C(5)), 1.63−1.52 (m, 2H, CH2CH2O), 1.28−1.23 (m, 29H, CH2-alk), 1.00 (d, J = 6.4 Hz, 1H, H−C(8′)), 0.89 (s, 10H, (CH3)3C−Si, H−C(8′)), 0.88−0.83 (m, 3H, CH3-alk), 0.18 (s, 3H, (CH3)2Si), 0.13 (s, 3H, (CH3)2Si); 13C NMR (101 MHz, CDCl3) δ 172.6 (CO2R), 164.2 (C(4)), 150.3 (C(2)), 136.4 (C(6)), 109.9 (C(5)), 92.9 (C(4′)), 88.8 (C(1′)), 85.4 (C(3′)), 67.2 (C(5′)), 65.1 (CH2O), 48.4 (C(2′)), 47.7 (C(7′)), 36.5 (C(9′)), 32.1 (C(6′)), 29.91, 29.87, 29.84, 29.81, 29.75, 29.6, 29.5 (CH2-alk), 28.8 (CH2CH2O), 26.1 (CH2-alk), 25.9 ((CH3)3C−Si), 22.9 (CH2-alk), 22.3 (C(8′)), 18.1 ((CH3)C-Si), 14.3 (CH3-alk), 12.8 (Me-C(5)), −3.7, −3.6 ((CH3)2Si); ESI+-HRMS m/z calcd for C37H65O7N2Si ([M + H]+) 677.4556, found 677.4556. 1-[(3′S,5′R,6′R)-6′-[(Hexadecoxycarbonyl)methyl]-2′-deoxy3′,5′-ethano-5′,6′-methano-β-D-ribofuranosyl]thymine (13). HF·pyridine (0.19 mL, 10.4 mmol) was slowly added at 0 °C to a solution of 12 (374 mg, 0.52 mmol) in pyridine (4 mL), and the mixture was left for 20 min before it was allowed to warm to rt. After 4575

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mL/min flow rate and detection at 260 nm. Purified oligonucleotides were desalted over Sep-Pak cartridges, quantified at 260 nm with a Nanodrop spectrophotometer, and analyzed by ESI− mass spectrometry. Oligonucleotides were then stored at −18 °C. UV−Melting Curves. UV−melting curves were recorded on a Varian Cary Bio100 UV/vis spectrophotometer. Absorbances were monitored at 260 nm, and the heating rate was set to 0.5 °C/min. A cooling−heating−cooling cycle in the temperature range 15−80 °C was applied. Tm values were obtained from the maximum of the first derivative curves and reported as the average of at least three ramps (±1 °C error). To avoid evaporation of the solution, the sample solutions were covered with a layer of dimethylpolysiloxane. All measurements were carried out in NaCl (150 mM), Na2HPO4 (10 mM) buffer at pH 7.0 with a duplex concentration of 2 μM. Fluorescence Spectroscopy. The solutions were prepared in NaCl (150 mM), Na2HPO4 (10 mM) buffer at pH 7.0 with a duplex concentration of 2 μM. Measurements were performed on a Varian Cary Eclipse fluorescence spectrophotomer in quartz cuvettes with a path length of 1 cm. The photomultiplier voltage was set to 500 V, and the solutions were excited at 480 nm. The emission spectra were recorded at 20 °C from 490 to 600 nm. Cell Culture and Transfection. HeLa and HEK 293T cells were grown at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 100 units/mL of penicillin and 100 μg/mL of streptomycin (P/S). Cells were split 1:10 every 2 or 3 days. For cellular uptake experiments, 1 × 105 HeLa and 2 × 105 HEK 293T cells were seeded in duplicate in six-well plates containing coverslips 24 h before treatment with oligonucleotides. Then, the medium was replaced by a 1 mL solution of fluoresceinlabeled oligonucleotide (10 μM final concentration) in DMEM + /+ (FCS, P/S). The medium was removed after 48 h at 37 °C and the cells were washed with 2 × 1 mL PBS. They were finally suspended in 1 mL fresh DMEM +/+. Cell Imaging. Fixation of the cells was carried out using a 1 mL solution of paraformaldehyde (3.7% PFA in PBS) for 10 min, after which the cells were washed with 2 × 1 mL PBS. After permeabilization of the cell membrane with 0.2% Triton x-100 for 10 min and washing with 2 × 1 mL PBS the cells were mounted on coverslips and treated with a few drops of polyvinyl alcohol (Mowiol) containing nuclear stain 4′,6′-diamidino-2-phenylindole (DAPI) and analyzed on a fluorescence microscope (Leica DMI6000 B, Leica Microsystems with Leica Application Suite software).

Data for 15: Rf = 0.77, 0.70 (EtOAc/hexane 7:3); 1H NMR (300 MHz, CDCl3) δ 8.31 (br, 1H, H-N(3)), 8.21−8.15 (m, 1H, H−C(6)), 7.47−7.38 (m, 2H, H-arom), 7.33 (dd, J = 8.8, 6.3 Hz, 4H, H-arom), 7.23−7.18 (m, 3H, H-arom), 6.76 (dd, J = 8.9, 7.8 Hz, 4H, H-arom), 5.83 (dd, J = 6.7, 4.4 Hz, 1H, H−C(1′)), 4.10−4.02 (m, 2H, CH2O), 3.79−3.75 (m, 6H, MeO), 3.71−3.37 (m, 4H, OCH2CH2CN, (Me2CH)2N), 2.86 (dd, J = 16.6, 3.2 Hz, 1H, H−C(9′)), 2.76−2.67 (m, 1H, H−C(2′)), 2.59−2.41 (m, 4.5H, OCH2CH2CN, H−C(9′), H−C(4′), H−C(7′)), 2.36−2.20 (m, 1.4H, H−C(7′), H−C(2′)), 2.12−2.08 (m, 3H, Me-C(5)), 2.07−2.00 (m, 1H, H−C(7′), H− C(2′)), 1.58−1.49 (m, 2H, CH2CH2O)), 1.32−1.16 (m, 26H, CH2alk), 1.12−1.03 (m, 12H, (Me2CH)2N), 0.94 (dd, J = 10.0, 6.5 Hz, 1H, H−C(8′)), 0.85 (t, J = 6.7 Hz, 3H, CH3-alk), 0.76 (d, J = 6.3 Hz, 1H, H−C(8′)); 13C NMR (101 MHz, CDCl3) δ 172.9, 172.7 (CO2R), 164.1 (C(4)), 159.04, 159.01 (MeO-C-arom), 150.2 (C(2)), 146.3, 146.2 (C-arom), 136.9, 136.8, 136.7 (C-arom, C(6)), 131.7, 131.6, 129.3, 127.7, 127.24, 127.17 (CH-arom), 117.8, 117.7 (CN), 112.94, 112.87 (CH-arom), 110.4, 110.3 (C(5)), 91.71, 91.67, 91.2, 91.1 (C(4′)), 88.5, 88.4, 88.3, 88.2, 88.1 (C(3′), C(Ph)3), 87.1, 86.8 (C(1′)), 68.4 (C(5′)), 65.0 (CH2O), 58.1, 58.0, 57.9, 57.8 (JC,P = 18.8 Hz, 19.5 Hz, OCH2CH2CN), 55.41, 55.38 (MeO), 47.2, 47.1 (C(2′)), 45.3, 45.1 (C(7′)), 43.6, 43.47, 43.45, 43.3 (JC,P = 12.9 Hz, 12.7 Hz, (Me2CH)2N), 36.7, 36.6 (C(9′)), 32.1 (C(6′)), 31.4, 31.3, 30.0, 29.9, 29.8, 29.7, 29.6, 29.5 (CH2-alk), 28.8 (CH2CH2O), 26.2 (CH2-alk), 24.7, 24.6, 24.54, 24.47, 24.4 ((Me2CH)2N), 22.9 (CH2-alk), 22.24, 22.20 (C(8′)), 20.51, 20.49, 20.44, 20.41 (JC,P = 8.0 Hz, 7.3 Hz, OCH2CH2CN), 14.33(CH3-alk), 12.7 (Me-C(5)). 31P NMR (162 MHz, CDCl 3 ) δ 143.7, 142.2; ESI + -HRMS m/z calcd for C61H86O10N4P ([M + H]+) 1065.6076, found 1065.6113. Oligonucleotide Synthesis. Syntheses of oligonucleotides were performed on the 1.3 μmol scale of a DNA synthesizer using standard solid-phase phosphoramidite chemistry. Oligomers were assembled using the manufacturer’s protocols on nucleoside preloaded CPG or fluorescein-labeled (FAM) solid supports (Roche Diagnostics). Natural phosphoramidites (dT, dC4Bz, dA6Bz,dG2dmf, Vivotide) were coupled as a 0.1 M solution in CH3CN, tricyclophosphoramidites (tcT, tc-C4Bz, tc-A6Bz, tc-G2dmf) as 0.15 M solution in CH3CN (0.15 M in dichloroethane for tc-A6Bz phosphoramidite). The coupling step was 90 s for natural phosphoramidites. An extended coupling time of 12 min for tricyclonucleosides was necessary to achieve average coupling efficiencies of 94% for tcee-T 10 and 97% for tchd-T 15 (trityl assay). As a coupling reagent, 5-(ethylthio)-1H-tetrazole (0.25 M in CH3CN) was used. Capping was performed with a solution of DMAP (0.5 M in CH3CN, Cap A) and a solution of 25% Ac2O and 12.5% sym-collidine in CH3CN (Cap B). Oxidation was performed with a solution of 20 mM I2 and 0.45 M sym-collidine in 2.1:1 CH3CN/H2O. Detritylation was carried out using a solution of 3% dichloroacetic acid in dichloroethane. Oligonucleotide Deprotection and Purification. Deprotection of the oligonucleotides containing amide-tc-T units (ON 1, 3, 5, 7−9), and detachment from the solid support was carried out using standard conditions (concd aq NH3 for 16 h at 55 °C). To maintain the ethyl and hexadecyl ester functions, ON 2, 4, 6, 12, and 13 were treated with a 1:3 NH3/EtOH solution at 40 °C for 24 h. ON 11 was deprotected in 25% benzylamine (EtOH/H2O 1:2) at 65 °C for 8 h. In all cases, the solutions were centrifuged after deprotection, the supernatants were removed and the remaining beads washed with 0.25 mL of H2O. The combined supernatants were then concentrated to dryness. To perform hydrolysis of the ethyl ester (ON 10), the solid phase was treated with a 0.1 M KOH solution. After an overnight shaking at 55 °C, the solution was neutralized with 1 M HCl and desalted (see below). Crude oligomers were purified by ion-exchange HPLC. As mobile phases, the following buffers were prepared: (A) 25 mM Trizma (2-amino-2-hydroxymethyl-1,3-propanediol) in H2O, pH 8.0; (B) 25 mM Trizma, 1.25 M NaCl in H2O, pH 8.0. For oligos containing tchd-T building blocks, ON 6: (A) 25 mM Trizma in H2O, pH 8.0; (B) 25 mM Trizma, 1 M NaClO4 in H2O, pH 8.0. ON 13: (A) 25 mM Trizma in H2O/ACN 4:1, pH 8.0; (B) 25 mM Trizma, 1 M NaClO4 in H2O/ACN 4:1, pH 8.0. Linear gradients of B in A were used (typically 0 to 40% or 0 to 50% B in A over 30 min), with a 1

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S Supporting Information *

1

H and 13C NMR spectra of all compounds, 31P NMR spectra of phosphoramidites 10 and 15. 1H−1H COSY and 2D NOESY spectra of compounds 3α and 4α, 1H NMR NOE spectrum and crystal data of compound 8. Characterization of oligonucleotides. Representative melting curves of ON 2, 4, 6, and 12−14. HPLC trace and mass spectrum of ON12 as a representative example. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*Phone: +41-31-631-4355. Fax: +41-31-631-3422. E-mail: [email protected].

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ACKNOWLEDGMENTS Financial support of this work by the Swiss National Science Foundation (Grant No. 200020-115913) and by the Association Monégasque contre les Myopathies (AMM) is gratefully acknowledged. We also thank the group of Chemical Crystallography of the University of Bern (PD Dr. P. Macchi) for the X-ray structure solution and the Swiss National Science Foundation (R’equip project 206021_128724) for cofunding 4576

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(31) Lavergne, T.; Baraguey, C.; Dupouy, C.; Parey, N.; Wuensche, W.; Sczakiel, G.; Vasseur, J.-J.; Debart, F. J. Org. Chem. 2011, 76, 5719−5731. (32) Parey, N.; Baraguey, C.; Vasseur, J.-J.; Debart, F. Org. Lett. 2006, 8, 3869−3872. (33) Luisier, S.; Leumann, C. J. Heterocycles 2010, 82, 775−790. (34) Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron 1968, 24, 53−58. (35) Staroscik, J. A.; Rickborn, B. J. Org. Chem. 1972, 37, 738−740. (36) Hoveyda, A. H.; Evans, D. A.; Fu, G. C. Chem. Rev. 1993, 93, 1307−1370. (37) Luisier, S.; Silhar, P.; Leumann, C. J. Nucleic Acids Symp. Ser. 2008, 52, 581−582. (38) Pallan, P. S.; Ittig, D.; Heroux, A.; Wawrzak, Z.; Leumann, C. J.; Egli, M. Chem. Commun. 2008, 883−885. (39) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984. (40) Cuppoletti, A.; Cho, Y.; Park, J.-S.; Strässler, C.; Kool, E. T. Bioconjugate Chem. 2005, 16, 528−534. (41) Ming, X.; Sato, K.; Juliano, R. L. J. Controlled Release 2011, 153, 83−92.

the single-crystal X-ray diffractometer at the department of Chemistry and Biochemistry of the University of Bern. We thank Prof. Oliver Mühlemann for providing access to the cell culture facility and equipment and Karin Schranz for technical assistance.

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